Atrial Fibrillation (AF) is the most common heart rhythm disorder (Benjamin E J, Levy D, Vaziri S M, D'Agostino R B, Belanger A J, Wolf P A. “Independent risk factors for atrial fibrillation in a population-based cohort. The Framingham Heart Study,” JAMA 1994; 271:840-4), and is a major risk factor for stroke and HF (Balasubramaniam R, Kistler P M. AF and “Heart failure: the chicken or the egg?” Heart 2009; 95:535-9; Lakshminarayan K, Anderson D C, Herzog C A, Qureshi A I. “Clinical epidemiology of atrial fibrillation and related cerebrovascular events in the United States,” Neurologist 2008; 14:143-50; Lip G Y, Kakar P, Watson T. “Atrial fibrillation—the growing epidemic” [comment], Heart 2007; 93:542-3). Since a majority of AF triggers arise in the pulmonary veins (PVs) and the adjoining posterior left atrium (PLA), ablation procedures that electrically isolate the PVs have emerged in recent years as a viable therapy for focal AF. Nonetheless, moderately high ablation success rates have only been achieved in selected patients (Nademanee K, McKenzie J, Kosar E et al. “A new approach for catheter ablation of atrial fibrillation: mapping of the electrophysiologic substrate” [see comment], J. Am. Coll. Cardiol. 2004; 43:2044-53; Nademanee K, Schwab M C, Kosar E M et al. “Clinical outcomes of catheter substrate ablation for high-risk patients with atrial fibrillation,” J. Am. Coll. Cardiol. 2008; 51:843-9; Taylor G W, Kay G N, Zheng X, Bishop S, Ideker R E. “Pathological effects of extensive radiofrequency energy applications in the pulmonary veins in dogs,” Circulation 2000; 101:1736-42). Indeed, in patients with structural heart disease e.g. heart failure (HF), success rates do not exceed 50-60% (Estner H L, Hessling G, Ndrepepa G et al. “Electrogram-guided substrate ablation with or without pulmonary vein isolation in patients with persistent atrial fibrillation,” Europace 2008; 10:1281-7; Weerasooriya R, Khairy P, Litalien J et al. “Catheter ablation for atrial fibrillation: are results maintained at 5 years of follow-up?” J. Am. Coll. Cardiol. 2011; 57:160-6). One reason for this low efficacy is that current ablation strategies primarily employ an anatomical, ‘one-size fits all’ strategy (with some minor variations) that does not address the specific mechanisms underlying AF (Ben Morrison T, Jared Bunch T, Gersh B J. “Pathophysiology of concomitant atrial fibrillation and heart failure: implications for management,” Nat. Clin. Pract. Cardiovasc. Med. 2009; 6:46-56). Recent research has therefore attempted to better define the mechanisms underlying AF, in order to improve upon the success of ablation and to develop new biological therapies for AF.
In the setting of structural heart disease—specifically HF—a variety of mechanisms e.g. stretch, oxidative stress (OS), autonomic imbalance and structural changes such as fibrosis are thought to contribute to a vulnerable AF substrate (Nattel S. “From guidelines to bench: implications of unresolved clinical issues for basic investigations of atrial fibrillation mechanisms,” Can. J. Cardiol. 2011; 27:19-26; Nattel S, Burstein B, Dobrev D. “Atrial remodeling and atrial fibrillation: mechanisms and implications,” Circ. Arrhythm. Electrophysiol. 2008; 1:62-73). OS is known to be elevated in the atria in AF (Youn J Y, Zhang J, Zhang Y et al. “Oxidative stress in atrial fibrillation: an emerging role of NADPH oxidase,” J. Mol. Cell. Cardiol. 2013; 62:72-9) and reactive oxygen species (ROS) have effects on the atrial action potential and Ca2+ cycling. However, the precise effects of ROS on atrial electrophysiology in the intact atria—and how these electrophysiological changes contribute to formation of AF substrate—are not known.
Oxygen derivatives with instabilities and increased reactivity, e.g. O2, H2O2, and OH, are generically termed ROS (Maejima Y, Kuroda J, Matsushima S, Ago T, Sadoshima J. “Regulation of myocardial growth and death by NADPH oxidase,” J. Mol. Cell. Cardiol. 2011; 50:408-16). While ROS at low doses mediates physiological functions such as growth, differentiation, metabolism (id.), excess ROS damages DNA, protein and lipids, and causes cell death (id.). A wealth of research points to increased OS as a key driver of cardiac remodeling caused by chronic pressure overload, loss of functional myocardium or AF (Kohlhaas M, Maack C. “Interplay of defective excitation-contraction coupling, energy starvation, and oxidative stress in heart failure,” Trends Cardiovasc. Med. 2011; 21:69-73; Maulik S K, Kumar S. “Oxidative stress and cardiac hypertrophy: a review,” Toxicol. Mech. Methods 2012; 22:359-66). In addition, chronic ROS elevation activates signaling pathways such as TGF-1, MAP kinase subfamilies (Hori M, Nishida K. “Oxidative stress and left ventricular remodelling after myocardial infarction,” Cardiovas. Res. 2009; 81:457-64; Tsai K H, Wang W J, Lin C W et al. “NADPH oxidase-derived superoxide anion-induced apoptosis is mediated via the JNK-dependent activation of NF-kappaB in cardiomyocytes exposed to high glucose,” J. Cell. Physiol. 2012; 227:1347-57) that result in structural changes e.g. fibrosis. Moreover, ROS generation (e.g. by Ang II mediated NOX activation) has been shown to lead to modifications of CaMKII (Erickson J R, He B J, Grumbach I M, Anderson M E. “CaMKII in the cardiovascular system: sensing redox states,” Physiol. Rev. 2011; 91:889-915), an important serine-threonine kinase involved in a variety of E-C coupling related processes in cardiac myocytes.
ROS are generated by the mitochondrial electron transport chain, the xanthine oxidase/dehydrogenase system, ‘uncoupled’ NOS, cytochrome P450 and NADPH oxidases. The NADPH oxidase enzyme family are a major source of cardiovascular ROS (Murdoch C E, Zhang M, Cave A C, Shah A M. “NADPH oxidase-dependent redox signalling in cardiac hypertrophy, remodelling and failure,” Cardiovasc. Res. 2006; 71:208-15; Cave A C, Brewer A C, Narayanapanicker A et al. “NADPH oxidases in cardiovascular health and disease,” Antioxid. Redox. Signal. 2006; 8:691-728) with NOX2 being the dominant ROS-generating NADPH isoform in HF (Nabeebaccus A, Zhang M, Shah A M. “NADPH oxidases and cardiac remodeling,” Heart Fail. Rev. 2011; 16:5-12; Maejima Y, Kuroda J, Matsushima S, Ago T, Sadoshima J. “Regulation of myocardial growth and death by NADPH oxidase,” J Mol. Cell. Cardiol. 2011; Cave A C, Brewer A C, Narayanapanicker A et al. “NADPH oxidases in cardiovascular health and disease,” Antioxid. Redox. Signal. 2006; 8:691-728; Zhang P, Hou M, Li Y et al. “NADPH oxidase contributes to coronary endothelial dysfunction in the failing heart,” Am. J. Physiol. Heart. Circ. Physiol. 2009; 296:H840-6; Dworakowski R, Alom-Ruiz S P, Shah A M. “NADPH oxidase-derived reactive oxygen species in the regulation of endothelial phenotype,” Pharmacol. Rep. 2008; 60:21-8). However, more recent studies indicate that NOX4 in mitochondria plays an essential role in mediating OS during pressure overload-induced cardiac hypertrophy (Nabeebaccus A, Zhang M, Shah A M. “NADPH oxidases and cardiac remodeling,” Heart Fail. Rev. 2011; 16:5-12; Kuroda J, Ago T, Matsushima S, Zhai P, Schneider M D, Sadoshima J. “NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart,” Proc. Natl. Acad. Sci. U.S.A. 2010; 107:15565-70; Zhang M, Brewer A C, Schroder K et al. “NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis,” Proc. Natl. Acad. Sci. U.S.A. 2010; 107:18121-6) NOX4 also appears to contribute significantly to the formation of fibrosis; in addition, it appears to be activated by pro-fibrotic signaling pathways e.g. TGF-β (Yeh Y H, Kuo C T, Chang G J, Qi X Y, Nattel S, Chen W J. “Nicotinamide adenine dinucleotide phosphate oxidase 4 mediates the differential responsiveness of atrial versus ventricular fibroblasts to transforming growth factor-beta,” Circ. Arrhythm. Electrophysiol. 2013; 6:790-8; Zhang M, Perino A, Ghigo A, Hirsch E, Shah A M. “NADPH oxidases in heart failure: poachers or gamekeepers?” Antioxid. Redox. Signal. 2013; 18:1024-41).
Recent evidence indicates that OS also contributes to structural and electrical remodeling in AF. Mihm et al. demonstrated significant oxidative damage in atrial appendages of AF patients undergoing the Maze procedure (Huang C X, Liu Y, Xia W F, Tang Y H, Huang H. Oxidative stress: a possible pathogenesis of atrial fibrillation. Med Hypotheses 2009; 72:466-7). Cames et al. showed that dogs with sustained AF had an increase in protein nitration, suggesting enhanced OS (Carnes C A, Janssen P M, Ruehr M L et al. “Atrial glutathione content, calcium current, and contractility,” J. Biol. Chem. 2007; 282:28063-73; Cames C A, Chung M K, Nakayama T et al. “Ascorbate attenuates atrial pacing-induced peroxynitrite formation and electrical remodeling and decreases the incidence of postoperative atrial fibrillation,” Circ. Res. 2001; 89:E32-8). Kim et al. showed that NADPH oxidase (NOX2) was a major source of atrial ROS in patients with AF (Kim Y M, Guzik T J, Zhang Y H et al. “A myocardial Nox2 containing NAD(P)H oxidase contributes to oxidative stress in human atrial fibrillation,” Circ. Res. 2005; 97:629-36). More recently, Reilly et al. have shown that atrial sources of ROS vary with the duration and substrate of AF, with NADPH oxidase being elevated early in AF (e.g. with post-operative AF) and with mitochondrial oxidases and uncoupled NOS being noted in long standing AF (Reilly S N, Jayaram R, Nahar K et al. “Atrial sources of reactive oxygen species vary with the duration and substrate of atrial fibrillation: implications for the antiarrhythmic effect of statins,” Circulation 2011; 124:1107-17). More recent data demonstrates that NOX4—which, as mentioned above, appears to contribute to the generation of mitochondrial ROS, specifically H2O2 (which more explicitly promotes fibrosis (Cucoranu I, Clempus R, Dikalova A et al. “NAD(P)H Oxidase 4 Mediates Transforming Growth Factor-1—Induced Differentiation of Cardiac Fibroblasts Into Myofibroblasts,” Circ. Res. 2005; 97:900-7))—is also elevated in AF (Joun et al. (2013) supra; Zhang J, Youn J Y, Kim A et al. “NOX4-dependent Hydrogen Peroxide Overproduction in Human Atrial Fibrillation and HL-1 Atrial Cells: Relationship to Hypertension,” Front. Physiol. 2012; 3)
The detrimental electrical effects of an enhanced, pathological late INa include the following: (i) diastolic depolarization during phase 4 of the AP that may lead to spontaneous AP firing and abnormal automaticity, (ii) an increase of AP duration, which may lead to EADs and triggered activity, as well as increased spatiotemporal differences of repolarization time, which promote reentrant electrical activity; and (iii) the indirect effects of a late INa-induced increase of Na+ entry to alter Ca2+ homeostasis in myocytes, which may lead to Ca2+ altemans and DADs. Acquired conditions and drugs that enhance late INa are associated with atrial tachyarrhythmias, ventricular tachyarrhythmias including torsades de pointes (TdF), afterpotentials (EADs, DADs), and triggered activity (Shryock J C, Song Y, Rajamani S, Antzelevitch C, Belardinelli L. “The arrhythmogenic consequences of increasing late INa in the cardiomyocyte,” Cardiovasc. Res. 2013; 99:600-11). Recent investigations implicate a role for abnormal Ca2+ handling in the genesis of ventricular and atrial arrhythmias (Aistrup G L, Balke C W, Wasserstrom J A. Arrhythmia triggers in heart failure: the smoking gun of [Ca2+]i dysregulation,” Heart Rhythm. 2011; 8:1804-8; Antoons G, Sipido K R. “Targeting calcium handling in arrhythmias,” Europace 2008; 10:1364-9; Laurita K R, Rosenbaum D S. “Mechanisms and potential therapeutic targets for ventricular arrhythmias associated with impaired cardiac calcium cycling,” J. Mol. Cell. Cardiol. 2008; 44:31-43). Abnormal Ca2+ handling can contribute to arrhythmogenesis directly by triggering abnormal depolarizations and indirectly by modulating action potential time course and duration. DADs are typically result from cellular Ca2+ overload, with SCR increasing forward NCX and producing an inward current resulting in DADs (Antoons et al. (2008) supra; Volders P G, Vos M A, Szabo B et al. “Progress in the understanding of cardiac early afterdepolarizations and torsades de pointes: time to revise current concepts,” Cardiovasc. Res. 2000; 46:376-92). More recent evidence has accumulated for Ca2+-mediated EADs (Volders et al. (2000) supra), which could contribute to triggered activity or at least prolong action potential duration. Indeed, abnormal Ca2+ cycling and resulting Ca2+ transient alternans predisposes to changes in the action potential that set up conditions for reentry (Laurita et al. (2008) supra). Abnormal SR Ca2+ release has also been suggested to contribute to reentry in the intact atria, including the PVs (Chou C C, Nihei M, Zhou S et al. “Intracellular calcium dynamics and anisotropic reentry in isolated canine pulmonary veins and left atrium,” Circulation 2005; 111:2889-97). In HF, a number of atrial ion-channel and E-C coupling proteins (Li D, Melnyk P, Feng J et al. “Effects of Experimental Heart Failure on Atrial Cellular and Ionic Electrophysiology,” Circulation 2000; 101:2631-8.61. Yeh Y-H, Wakili R, Qi X-Y et al. “Calcium-Handling Abnormalities Underlying Atrial Arrhythmogenesis and Contractile Dysfunction in Dogs With Congestive Heart Failure,” Circ. Arrhythm. Electrophysiol. 2008; 1:93-102) can be modulated by ROS (Hool L C. “Reactive Oxygen Species in Cardiac Signalling: From Mitochondria to Plasma Membrane Ion Channels,” Clin. Exp. Pharm. Phys. 2006; 33:146-51; Zima A V, Blatter L A. “Redox regulation of cardiac calcium channels and transporters,” Cardiovasc. Res. 2006; 71:310-21; Nediani C, Raimondi L, Borchi E, Cerbai E. “Nitric Oxide/Reactive Oxygen Species Generation and Nitroso/Redox Imbalance in Heart Failure: From Molecular Mechanisms to Therapeutic Implications,” Antioxidants & Redox Signaling 2011; 14:289-331) at least in part via ROS activation of kinases and inactivation of phosphatases, resulting in aberrant phosphorylation (e.g. of RyR2 and phospholamban). Also, ROS directly decrease SERCA function, but increase NCX function (Koster G M, Lancet S, Zhang J et al. “Redox-mediated reciprocal regulation of SERCA and Na+—Ca2+ exchanger contributes to sarcoplasmic reticulum Ca2+ depletion in cardiac myocytes,” Free Rad. Biol. Med. 2010; 48:1182-7) which parallels the changes in SERCA and NCX in HF. Additionally, ROS increases late/persistent INa(INa_late) (Luo A, Ma J, Zhang P, Zhou H, Wang W. “Sodium Channel Gating Modes During Redox Reaction,” Cell. Phys. Bioch. 2007; 19:9-20), which again parallels that in HF (Valdivia C R, Chu W W, Pu J et al. “Increased late sodium current in myocytes from a canine heart failure model and from failing human heart,” J. Mol. Cell. Cardiol. 2005; 38:475-83) and INa, late can significantly contribute to the induction of EADs and DADs (Li D, Melnyk P, Feng J et al. “Effects of Experimental Heart Failure on Atrial Cellular and Ionic Electrophysiology,” Circulation 2000; 101:2631-8; Song Y, Shryock J C, Belardinelli L. “An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes,” Am. J. Physiol.—Heart and Circ. Physiol. 2008; 294:H2031-H9; Wasserstrom J A, Sharma R, O'Toole M J et al. “Ranolazine Antagonizes the Effects of Increased Late Sodium Current on Intracellular Calcium Cycling in Rat Isolated Intact Heart,” J. Pharm. Exp. Ther. 2009; Undrovinas N, Maltsev V, Belardinelli L, Sabbah H, Undrovinas A. “Late sodium current contributes to diastolic cell Ca&lt;sup&gt;2+&lt;/sup&gt; accumulation in chronic heart failure,” J. Physiol. Sci. 2010; 60:245-57) both in ventricles and atria. Additional promotion of triggered activity could come from the increased Ca2+ sensitivity of hyperphosphorylated RyR2s in HF (Terentyev D, Gyorke I, Belevych A E et al. “Redox Modification of Ryanodine Receptors Contributes to Sarcoplasmic Reticulum Ca2+ Leak in Chronic Heart Failure,” Circ. Res. 2008; 103:1466-72), which together with OS modifications of RyR2 (nitrosylation, oxidation) (Valdivia C R, Chu W W, Pu J et al. “Increased late sodium current in myocytes from a canine heart failure model and from failing human heart,” J. Mol. Cell. Card. 2005; 38:475-83. 65. Song Y, Shryock J C, Belardinelli L. “An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes,” Am. J. Physiol. Heart Circ. Physiol. 2008; 294:H2031-H9) in HF lead to leaky ventricular RyR2s (Gonzalez D R, Beigi F, Treuer A V, Hare J M. “Deficient ryanodine receptor 5-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes,” Proc. Natl. Acad. Sci. U.S.A. 2007; 104:20612-7; Marx S O, Marks A R. “Dysfunctional ryanodine receptors in the heart: new insights into complex cardiovascular diseases,” J. Mol. Cell. Cardiol. 2013; 58:225-31). The oxidation/nitrosylation state of atrial RyR2s in HF has not been fully scrutinized; however, there appear to be significant differences in atrial versus ventricular E-C coupling, as has previously been suggested by others (Bootman M D, Smymias I, Thul R, Coombes S, Roderick H L. “Atrial cardiomyocyte calcium signaling,” Biochim. Biophys. Acta 2011; 1813:922-34). The majority of the studies mentioned above have been performed in isolated myocytes; the specific contribution oxidized INa, RyR2 to the electrophysiological characteristics of the intact atrium—and how this contributes to arrhythmogenesis—is not known.
Excessively activated CaMKII is implicated in the genesis of HF and arrhythmias. Recent evidence suggests that both CaMKII and H2O2 increase RyR2 (Marx S O, Marks A R. “Dysfunctional ryanodine receptors in the heart: new insights into complex cardiovascular diseases,” J. Mol. Cell. Cardiol. 2013; 58:225-31; Niggli E, Ullrich N D, Gutierrez D, Kyrychenko S, Polakova E, Shirokova N. “Posttranslational modifications of cardiac ryanodine receptors: Ca(2+) signaling and EC-coupling,” Biochim. Biophys. Acta 2013; 1833:866-75) PO (Undrovinas N, Maltsev V, Belardinelli L, Sabbah H, Undrovinas A. “Late sodium current contributes to diastolic cell Ca&lt;sup&gt;2+&lt;/sup&gt; accumulation in chronic heart failure,” J. Physiol. Sci. 2010; 60:245-57; Gonzalez D R, Beigi F, Treuer A V, Hare J M. “Deficient ryanodine receptor S-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes,” Proc. Natl. Acad. Sci. U.S.A. 2007; 104:20612-7; Donoso P, Sanchez G, Bull R, Hidalgo C. “Modulation of cardiac ryanodine receptor activity by ROS and RNS,” Front. Biosci. (Landmark Ed) 2011; 16:553-67; Terentyev D, Gyorke I, Belevych A E, et al. “Redox modification of ryanodine receptors contributes to sarcoplasmic reticulum Ca2+ leak in chronic heart failure,” Circ. Res. 2008; 103:1466-72), thereby promoting SR Ca2 leak and arrhythmias (Belevych A E, Terentyev D, Terentyeva R et al. “Shortened Ca2+ signaling refractoriness underlies cellular arrhythmogenesis in a postinfarction model of sudden cardiac death,” Circ. Res. 2012; 110:569-77). Since OS-mediated oxidation of Met 281/282 residues in the regulatory domain of CaMKII transforms CaMKII into a constitutively active form (ox-CaMKII), leading to aberrant phosphorylation of multiple E-C coupling proteins (Swaminathan P D, Purohit A, Hund T J, Anderson M E. “Calmodulin-dependent protein kinase II: linking heart failure and arrhythmias,” Circ. Res. 2012; 110:1661-77; Erickson J R, He B J, Grumbach I M, Anderson M E. “CaMKII in the cardiovascular system: sensing redox states,” Physiol. Rev. 2011; 91:889-915), OS may affect RyR2PO both directly (via ROS) and indirectly (via ox-CaMKII). Indeed, expression of ox-CaMKII has been found to be increased in atria of AF patients, indicating a potential role of ROS induced CaMKII activation in AF (Purohit A, Rokita A G, Guan X et al. “Oxidized Ca2+/Calmodulin-Dependent Protein Kinase II Triggers Atrial Fibrillation,” Circulation 2013; 128:1748-57). CaMKII has also been shown to modulate the gating of NaV1.5, at least in part by phosphorylation of NaV1.5 at multiple sites (Ashpole N M, Herren A W, Ginsburg K S et al. “Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites,” J. Biol. Chem. 2012; 287:19856-69). A recent study demonstrates a fundamental requirement for targeting of CaMKII to a controlling phosphorylation site, 5571, on NaV1.5 (Hund T J, Koval O M, Li J et al. “A beta(IV)-spectrin/CaMKII signaling complex is essential for membrane excitability in mice,” J. Clin. Invest. 2010; 120:3508-19).
CaMKII phosphorylation of NaV1.5 is thought to decrease transient INa, but increase INa,late, again thereby contributing to the genesis of triggered activity (Hashambhoy Y L, Winslow R L, Greenstein J L. “CaMKII-dependent activation of late INa contributes to cellular arrhythmia in a model of the cardiac myocyte,” Conf. Proc. IEEE Eng. Med. Biol. Soc. 2011; 2011:4665-8). Modeling studies also suggest that ox-CaMKII may create substrate for reentry by regulating conduction characteristics of the myocardium (Hashambhoy et al. (2011) supra; Christensen M D, Dun W, Boyden P A, Anderson M E, Mohler P J, Hund T J. “Oxidized calmodulin kinase II regulates conduction following myocardial infarction: a computational analysis,” PLoS Comput. Biol. 2009; 5:e1000583), with this substrate thought to be at least partially mediated by modulation of INa.
A summary of the various mechanisms for reactive oxygen species production, oxidative stress generation, and development of fibrosis and atrial fibrillation is illustrated in FIG. 1.